The pathological hallmarks of Alzheimer's Disease (AD) are the insoluble neuritic plaques composed primarily of amyloid-beta (Aβ).[1] Aβ is a 37-43 amino acid peptide associated with a variety of neurotoxic events ranging from diminished integrity of the blood-brain barrier due to oxidized lipids of endothelial cell membranes, to accumulation of hyperphosphorylated tau leading to damaged synapses and neuronal cell death[2] Evidence of Aβ toxicity is widespread in the post-mortem AD brain and thus limiting its toxicity and preventing its accumulation have become popular goals of medicinal research.[3]
Production of Aβ results from sequential cleavage of a transmembrane protein called amyloid precursor protein (APP) by two proteases. β-secretase (BACE) is a membrane-associated aspartic protease that cleaves APP in the extracellular domain and creates the N-terminus of Aβ.[4] Subsequent cleavage by γ-secretase in the membrane domain creates the C-terminus and releases the toxic Aβ fragment.[5] While this process is normal for most cells[6] it is actually a minor APP processing pathway. The primary metabolic fate of APP is initial hydrolysis by α-secretase in the middle of the Aβ domain, thus precluding Aβ formation, followed by γ-secretase processing to release protein fragments from the membrane.[7] In contrast to Aβ however, the peptides resulting from α-secretase cleavage appear to have neuroprotective effects[8] Further implicating BACE as a facilitator of AD are observations that genetic mutations that either increase BACE cleavage or decrease α-secretase activity result in increased Aβ production and early-onset AD.[9] Conversely, experiments that inhibit BACE activity in mice by either gene knockdown, interfering RNA, or APP mutation, have proven to reduce amyloid plaque loads and restore cognitive abilities.[4] These results have led to an increased effort to design BACE inhibitors as therapeutic agents for AD.
Developing potent and specific BACE inhibitors has been challenging due to the broad substrate specificity and large active site of the enzyme.[10] A popular assay used to monitor inhibitor efficacy in vitro utilizes a FRET substrate that increases its fluorescence emission when hydrolyzed by BACE. Effective BACE inhibitors therefore suppress the fluorescence turn-on effect. This approach remains the standard in assaying in vitro BACE activity[11] and in fact, the reagents for this fluorescence assay are commonly sold together as kits by multiple chemical suppliers. Because this technique is amenable to high-throughput screening, it has been used extensively to identify BACE inhibitors.[12] Many of these seemingly promising inhibitors, however, are disappointingly inadequate at inhibiting BACE activity when assayed in cell models.
The failure in cellular activity of inhibitors is due in part to the cellular localization of active BACE enzyme. Even though BACE is expressed on the extracellular membrane of cells, it is inactive at pH 7.4 and thus does not cleave APP when exposed to the extracellular environment. BACE must be endocytosed to an early endosome where ATPase pumps protons into the endosomal lumen and lowers the pH to between 4-5 in order to gain activity.[13] Thus, inhibitors that do not specifically access these endosomal compartments are unable to inhibit BACE in cellular models. Add in challenges with bioavailability, metabolic clearance, and brain access, and it is clear why designing potent BACE inhibitors has been extremely difficult.[14]
Compounding design problems are the assays used to evaluate inhibitors in cells. The typical ELISA assays for determining cellular efficacy of these inhibitors are slow, expensive, and laborious. They often require genetic manipulations of the cell lines, several costly antibodies, and take multiple days.[15] An alternative FRET-based assay has been developed, but still requires genetic manipulation and cannot easily detect real-time fluorescence changes.[16]
Accordingly, there remains an unmet need for molecular probes to image real-time BACE activity in living cells and organisms that do not require the use of mutated cell lines or antibodies. The presently disclosed subject matter provides such BACE probes.